The present invention relates to method and apparatus for concentrating fluid-borne pathogens from fluids potentially containing such pathogens. More specifically, the present invention relates to methods and apparatus for concentrating pathogens in a centrifugal chamber and for re-suspending them to permit withdrawal of the concentrated pathogens from the chamber for testing, quantifying and the like.
It is known to use centrifuges for the purpose of concentrating water and food-borne microorganisms, particularly pathogens including Clostridium, Streptococcus, Shigella, Salmonella, and other species, as set forth in U.S. Pat. Nos. 5,961,846; 5,858,251; and 5,846,439, all of which are hereby incorporated by reference into this description. These patents disclose a technique for flowing large quantities of water or fluidized foods through a semi-rigid belt channel in a blood centrifuge, such as the IBM Model 2997 or the Cobe Spectra centrifuge, to concentrate any microorganisms contained in the fluid.
These centrifuges employ a disposable annular or circumferential separation chamber that is mounted on a reusable hardware platform. The centrifuge rotates the separation channels as fluid flows through the channel, concentrating microorganisms within the channel. In order to test, identify or otherwise evaluate any pathogens or other microorganisms concentrated in the channel at the end of the process, the disposable channel must be removed from the centrifuge device, and any pathogens or other microorganisms contained therein must be flushed from the channel.
Although the centrifuges may work satisfactorily for concentrating fluid-borne microorganisms, the steps of re-suspending and removing concentrated pathogens from the centrifuge separation channel have presented some difficulty, and the above-identified patents describe a relatively complex technique for recovery of the channel contents after the centrifugation process has ended. First, according to the ""439 patent, the separation channel is primed with water containing a surfactant to enhance removal of the material later collected. In addition, after the centrifugation is completed, and the contents of the separation channel are drained into a beaker, the channel is then cut in half and filled with a solution of surfactant. The cut ends are clamped with Vise-Grip(copyright) pliers, and the channel is shaken vigorously and placed in a laboratory vortex to dislodge pathogens that may have adhered to the inner walls of the channel. This rinsing procedure is conducted several times, and the concentrate and all the rinses are combined.
The disposable centrifuge channels used in the IBM 2997 and COBE Spectra centrifuges are made of semi-rigid, somewhat brittle, plastic material, which is not conducive to repeated flexing or the like. This may have contributed to the difficulty in removing concentrated pathogens from the channel and necessitated the use of surfactant, Vise-Grip pliers and a laboratory vortex to aid in removing the concentrated microorganisms. Also, the presence of other residue in the channel may have made removal of the microorganisms more difficult. The present invention is intended to overcome one or more of the shortcomings associated with the prior art devices and methods. As used in the following description and claims, xe2x80x9cfluidxe2x80x9d (and formatives thereof) means any liquid, excluding blood, blood cells, plasma or other blood components, that flows sufficiently for continuous centrifugal processing, and xe2x80x9cpathogensxe2x80x9d and xe2x80x9cpathogenic organismsxe2x80x9d (and formatives thereof) mean a disease-causing or abnormality-causing organism and do not include, in any event, blood cells such as red cells, white cells and platelets.
The present invention is generally embodied in method and apparatus for concentrating and recovering pathogens from fluid by employing a flexible centrifugation chamber, through which the fluid is continuously flowed. The flexible centrifugation chamber is subjected to centrifugal force by rotating the chamber about an axis of rotation while fluid is being fed therethrough, so as to concentrate in the chamber pathogens that may be contained in the fluid. In accordance with the present invention, the flexibility of the chamber enhances re-suspension of pathogens that are concentrated therewithin, and the pathogens may be re-suspended by shaking the flexible chamber with fluid contained therein. By vigorously shaking the container to and fro, the fluid therein is caused to slosh from end to end by virtue of the flexibility of the chamber. This induces high shear stresses and promotes re-suspension of the pathogens. The flexible chamber may also be stretched such that the gap of the chamber can be adjusted. By doing this, one can induce and control proper shear stress. This can be done manually or automatically.
The step of shaking the chamber may be carried out manually or automatically and may include squeezing and/or twisting of the chamber to cause the fluid to slosh back and forth.
The flexible centrifugation chamber may be elongated, and fluid may be introduced into the chamber substantially at one end and withdrawn substantially at the other end of chamber. Alternatively or additionally, the chamber may be subdivided into a series of interconnected flow channel segments so that fluid repeatedly substantially traverses the length or width of the container as it passes therethrough, thereby decreasing stagnation or unperfused areas of the chamber and resulting in a more uniform flow field in the centrifugal field, and thus enhancing concentration of pathogens in the chamber. Other serpentine flow path arrangements may also be used within the flexible chamber.
In accordance with the present invention, a single centrifuge chamber may be used in the concentration procedure. Also, multiple chambers, formed of entirely separate chambers or formed from a single disposable unit or chamber sub-divided into two or more subchambers, may be employed for higher fluid processing rates or collection efficiency. For example, the use of separate containers or sub-chambers with separate inlets connected in parallel to the fluid source may allow for higher processing rates, since fluid is simultaneously being processing through two chambers.
Also, separate chambers or sub-chambers may be connected in series for improved efficiencies. The second chamber could be used for the more specific collection of pathogens from the fluid. In other words, the supernatant from the first stage will include many of the target pathogens which can be concentrated in the second chamber or stage. For particularly small pathogens, a sedimentation enhancing agent, such as an affinity agent, for example, a chemical enzyme, may be added to the supernatant from the first chamber to enhance sedimentation of the pathogenic organisms contained therein.
In a multiple stage or multiple chamber separation, the first chamber in the series could be a simple plastic pouch, with or without a simple u-shaped or other flow path, for collection of a large volume of sediment. The second container could employ the same or a lengthier flow path, such as shown in FIG. 4a or 4b. In either the parallel or series arrangement, one chamber (container) could immediately be used for testing, identifying or quantifying the pathogens, and the other chamber could be severed, sealed and stored as an archive for future testing or reference if desired. Additional chambers (more than two) also could be employed in parallel or series in accordance with this aspect of the present invention. Also, in the series arrangement, the first chamber could include a passageway for withdrawal of concentrated particles (which may include some of the pathogens) on an intermittent or continuous basis.
The flexible centrifugation chamber may be fashioned in various different ways without departing from the present invention. In one preferred embodiment, the chamber is defined by a pair of facing sheets of flexible plastic film that are sealed together, as by heat or solvent, along at least a peripheral area to define an interior chamber for centrifugal fluid processing. Other forming techniques may also be used, provided that the end result is a flexible centrifugation chamber that may be easily deformed for re-suspension. For example, a rigid or semi-rigid chamber could be used with a flexible liner. The rigid or semi-rigid container could provide the desired shape for centrifugation purposes, and the flexible liner removed after centrifugation for easy re-suspension. Also, the chamber could be partially rigid or semi-rigid and partially flexible. The areas of the chambers where the pathogens concentrate could be made flexible, and the remainder of the chamber or container could be rigid or semi-rigid, which may be easier to shake.
In addition to the peripheral seal, other seal lines may be provided between the facing plastic sheets to define an elongated or serpentine flow path or to define a plurality of interconnected flow channel segments within the chamber to potentially improve the uniformity of the flow fields of fluid passing through the chamber and enhance the collection efficiency. These additional seal lines may be provided permanently by bonding together the facing plastic sheets, as by heat or solvent bonding, or may be provided temporarily by compressing the plastic sheets together in the desired locations to form the desired flow path configuration during centrifugation and allowing the films to separate to form a single chamber after centrifugation is complete. These are but a few of the features of the present invention found in the following more detailed description.